This invention is generally in the field of blood and specifically in the area of platelet proteins.
Blood platelets are the terminal differentiation product of megakaryocytes. Megakaryocytes are believed to originate from the primordial pluripotential stem cells of the bone marrow. The earliest recognizable form is the megakaryoblast which possesses an immature nucleus embedded in basophilic cytoplasm with a minimal number of granules. The complex maturation process involves the formation of a multilobulated, polyploid nucleus and distinctive, highly specialized cytoplasmic granules. Platelets are shed from mature megakaryocytes by a process that is poorly understood.
Although progress has been made in identifying the synthesis of platelet-specific proteins and subcellular structure in maturing megakaryocytes, relatively little is known about the regulation of megakaryocytopoiesis.
Several humoral factors have been postulated to control the ploidization and cytoplasmic maturation of megakaryocytes. These substances have been obtained in crude form and are termed thrombopoietin (TPO) or thrombopoietic stimulatory activity (TSF), depending upon the source from which the activity is derived. The assays used to quantitate the biological activity of these factors are dependent upon measurements of the incorporation of radioisotopes, especially [.sup.75 Se]selenomethionine and .sup.35 SO.sub.4, into the proteins and glycosaminoglycans of newly-formed platelets, all of which are synthesized by the megakaryocytes. Accordingly, these measurements probably reflect the rates of cytoplasmic maturation of megakaryocytes including the development of the specialized cytoplasmic structures integral to platelet function.
Recent data indicates that a megakaryocyte-colony stimulating factor (Meg-CSF) causes the tremendous increase in DNA replications. The second phase, the maturation of the committed precursor cells to fully differentiated megakaryocytes, is thought to be regulated by a humoral factor(s) whose blood concentration is somehow altered by changes in the level of circulating platelets.
This factor(s) increases the transcription rate of proteins for inclusion into multiple secretory granules. The cytoplasm of the mature megakaryocyte contains multiple platelet secretory granules, including alpha-granules, dense granules, and platelet lysosomes. In particular, the alpha-granules are known to contain platelet-derived growth factor (PDGF), beta-thromboglobulin, platelet factor 4 (PF4), and the chondroitin sulfate proteoglycan carrier.
Previous investigators have described procedures for isolating a partially purified thrombopoietically active substance(s), including T. P. McDonald et al. in Exp. Hematol. 9, 288-296 (1981), J. Levin et al. in Blood 54, 166a (abst.) (1979), and T. P. McDonald in Biochem. Med. 21, 146-155 (1979). These partially purified preparations, approximately 100 fold purified, were generally prepared by ammonium sulfate precipitation and chromatography on a lectin column. Although they were shown to have some role in stimulating protein synthesis, no single factor in pure form has been isolated for characterization and application.
It is therefore an object of the present invention to identify and isolate a factor regulating maturation of committed megakaryocyte progenitor cells to fully differentiated megakaryocytes, particularly a factor enhancing the rate of protein synthesis.
It is a further object of the present invention to provide a method for isolation of very pure megakaryocyte stimulatory factor.
It is still a further object of the invention to provide methods for specifically assaying for megakaryocyte stimulatory factor.
It is another object of the present invention to provide nucleotide sequences encoding megakaryocyte stimulatory factor proteins.